Cloning and Expression of In silico Modeled Protein Enriched with Branched Chain Amino Acids in Pichia pastoris.

Sunil, L. and Prasanna, Vasu (2020) Cloning and Expression of In silico Modeled Protein Enriched with Branched Chain Amino Acids in Pichia pastoris. International Journal of Biological Macromolecules, 146. pp. 739-745. ISSN 0141-8130

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Abstract

We have earlier in silico designed the 3-dimensional structure of a protein enriched with branched chain amino acids (BCAA, 56.4%), having only α-helical coiled-coil structure. Here, homology modeling was used to improve the in silico designed protein model. The secondary and tertiary structures of improved protein model were predicted, and validated using various online bioinformatics tools. The amino acid sequence of the final predicted Protein Model-51 was EQLTKLEIVIRVLKLLKLIGGLVSLVEWVLTALVTLLGDKVLDDILTDVIMLVKKIL DKVIGIVYVLAILALILSEVLDILWLLEKLVEILEGHHHHHH. The amino acid sequence of the protein model was reverse translated to DNA sequence and codons were optimized using codon optimization tool. The chemically synthesized BCAA51 gene was cloned to pPICZαC vector, and transformed into DH5α E. coli strain. After successful transformation, the protein was expressed in P. pastoris system by inducing with 0.5% methanol, every 24 h for up to 144 h. The expressed protein was purified by His Select Nickel affinity chromatography with an yield of 1.412 mg/L. The recombinant protein was confirmed by SDS-PAGE and western blot analysis, which showed a clear band at the expected molecular weight of ~11 kDa. Thus, here we have shown that the in silico designed protein is successfully cloned and expressed in P. pastoris.

Item Type:	Article
Uncontrolled Keywords:	BCAA modeled protein; Branched chain amino acids; P. pastoris
Subjects:	600 Technology > 08 Food technology > 16 Nutritive value > 03 Proteins 600 Technology > 08 Food technology > 29 Microbiological food > 04 Yeast
Divisions:	Food Safety Analytical Quality Control Lab
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	27 Apr 2020 06:04
Last Modified:	27 Apr 2020 06:04
URI:	http://ir.cftri.res.in/id/eprint/14261

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